Browsing by Author "Hunter, Charles Haig."

Now showing 1 - 20 of 20

An assessment of synthetic landfill leachate attenuation in soil and the spatial and temporal implications of the leachate on bacterial community diversity.
(2008) Govender, Kamenthren.; Hunter, Charles Haig.
The temporal fate of selected parameters, including redox potential; pH; phenol; nitrates; sulphates; copper and zinc, of a young synthetic acetogenic phase landfill leachate was assessed by perfusing a series of sequential soil (Hutton) microcosms (arrays) at two hydraulic loading rates (HLR). We chose HLRs that were representative of areas in South Africa with typically elevated rainfall (Pietermaritzburg – HLRh) and one with relatively low rainfall (Kimberley – HLRl). Preliminary phenol, copper, and zinc adsorption investigations on gamma radiation sterilized soil and unsterilized soil revealed superior adsorption rates for each compound in the unsterilized soil. This revealed the importance of the biological component of soil in phenol, copper, and zinc attenuation in soil. The results presented in this thesis suggest that the HLR of leachate into soil arrays contributes to significant differences in the fate of the landfill leachate parameters mentioned earlier. In addition, we assessed the temporal and spatial succession of bacterial community diversity in each of the soil arrays by polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). Prior to this, we compared two soil DNA isolation techniques, the modified method of Duarte et al. (1998) (Bead Beat) and the commercial Mo-Bio UltraClean™ Soil DNA isolation kit (Kit). The DNA isolated by the Kit method was significantly superior regarding purity and absence of DNA fragmentation. However, the Bead Beat method produced a significantly higher yield per reaction before further purification with Wizard™ Clean-Up columns produced DNA extracts of similar purity at the cost of a significant reduction in DNA yield. The Kit method was chosen for future DNA isolation and PCR-DGGE based on the quality of the PCR amplicons generated from the Kit isolated DNA. PCR-DGGE was further optimized by comparing the efficiency and sensitivity of a silver stain against ethidium bromide. Silver stain generated DGGE gels with greater number of bands (species richness – S) and stronger band signal intensities. Captured DGGE fingerprints generated data that were subjected to the Shannon-Weaver Index (H’) and the associated Shannon-Weaver Evenness Index (EH) to measure the change in spatial and temporal bacterial diversity. There was a significant shift in S and H’ for both HLRs but a significant change in EH was only observed for HLRh. Furthermore, a temporal comparison of S and H’ between both HLRs revealed significant differences throughout the investigation. Canonical Correspondence Analysis (CCA) revealed spatial distribution of bacterial community diversity with depth. Effects of phenol concentration, redox potential, and pH of the effluent leachate on bacterial community diversity was tentatively assessed by three-dimensional graphical representation on PlotIT 3.2 software. Bacterial community diversity showed a decrease with elevated pH and phenol concentration along with decreasing redox potentials for both HLRs. While this study reveals the spatial and temporal dynamics of bacterial community diversity in situ, it provides important evidence with respect to: (i) the effects of rainfall / leaching rates (HLR) on spatial and temporal bacterial community succession; (ii) the importance of the biological component in natural attenuation; (iii) the ability of soil, previously unexposed to landfill leachate, to initiate natural attenuation of phenol and other leachate constituents; (iv) the capacity of PCRDGGE to fingerprint successional changes in bacterial community diversity, (v) and the potential to clone and sequence selected members of bacterial associations for future reference in environmental remediation strategies.
Biochemical and microbiological changes in sugarcane stalks during a simulated harvest-to-crush delay.
(2008) Martin, Lauren Anne.; Hunter, Charles Haig.; Watt, Derek Alexander.
Post-harvest cane deterioration in the South African sugar industry results in significant revenue loss that is estimated to be in the region of ZAR 60 million per annum. Despite these large losses, precise biological data relating to the process of cane deterioration under South African conditions is limited. Severity of deterioration is influenced by a number of factors, including the length of the harvest-to-crush delay (HTCD), ambient temperature and harvesting practices. For example, burning of cane prior to harvest may result in rind splitting, which provides entry for microbes, particularly Leuconostoc mesenteroides that may exacerbate deterioration. The effect of these factors on deterioration was examined by quantifying the biochemical and microbiological changes that occur in sugarcane stalks after harvest, with the influence of length of HTCD, degree of L. mesenteroides infection and ambient temperature receiving attention. The primary novelty of the work resides in the analysis of deterioration under tightly regulated temperatures, which were designed to reflect diurnal variations typically experienced during summer and winter in the South African sugar belt. In addition, inoculation of mature internodes with a consistent titre of L. mesenteroides was used as a means to mimic a consistent level of infection of harvested stalks by the bacterium. Metabolites selected for analysis were those both native to the stalk and produced as by-products of microbial metabolism, viz. sucrose, glucose, fructose, ethanol, lactic acid, dextran and mannitol. Simulated HTCDs under summer temperatures resulted in increasing glucose and fructose levels with time, which contrasted to the approximately constant levels of these hexose sugars under winter conditions. Commonly referred to as ‘purity’ in an industrial context, precise determination of the concentration of these hexoses in cane consignments could potentially indicate the extent of deterioration. Despite the detection of a basal concentration of lactic acid in unspoiled cane, the observed increase in concentration of this organic acid over the simulated summer HTCD suggests that this metabolite could also potentially serve as an indicator for postharvest deterioration. In contrast, the investigation indicated that ethanol was an unsuitable biochemical marker for deterioration of L. mesenteroides infected cane. An inability to detect dextran and mannitol in the samples, combined with consistent sucrose levels and variable mill room data, suggest that extreme proliferation of L. mesenteroides is facilitated primarily by in-field practices, particularly the manner in which cane is prepared prior to harvest and transport to the mill. Bacterial proliferation and infection by L. mesenteroides of inoculated stalks were monitored by standard selective culturing techniques. Despite the limited detection of L. mesenteroides-associated metabolites, culture-based analyses revealed that the bacterium was the dominant bacterial species within the samples. A number of other bacterial species were isolated and identified, however the extent to which the total number of microorganisms proliferated was limited to a maximum of 1 x 105 colony forming units per gram of fresh tissue. In conjunction with these analyses, a molecular approach known as Polymerase Chain Reaction-Mediated Denaturing Gradient Gel Electrophoresis (PCR-DGGE) was undertaken to investigate the bacterial diversity patterns associated with deteriorating sugarcane stalks throughout the delay period. In contrast to the results obtained by means of the culture-based assays, PCR-DGGE revealed that L. mesenteroides was not the dominant bacterial population, and showed that the level of bacterial diversity was relatively consistent across the differing treatments and with time. The use of complimentary culture-dependent and cultureindependent analyses thus permitted the detection of this discrepancy and indicated the utility of PCR-DGGE in the determination of bacterial community structure of postharvest sugarcane tissue. The biology of post-harvest deterioration of green sugarcane stalks is highly complex, even under rigorously controlled temperature and infection regimens. The results of this study emphasize the important effects that harvest method and environmental conditions have on post-harvest sugarcane deterioration. Towards the formulation of industry-relevant recommendations for combating post-harvest deterioration, future work will strive to mimic the effects that harsh harvesting and transport practices have on the severity of the problem.
Biological control and plant growth promotion by selected trichoderma and Bacillus species.
(2005) Yobo, Kwasi Sackey.; Laing, Mark Delmege.; Hunter, Charles Haig.
Various Trichoderma and Bacillus spp. have been documented as being antagonistic to a wide range of soilborne plant pathogens, as well as being plant growth stimulants. Successes in biological control and plant growth promotion research has led to the development of various Trichoderma and Bacillus products, which are available commercially. This study was conducted to evaluate the effect of six Trichoderma spp. and three Bacillus spp. and their respective combinations, for the biological control of Rhizoctonia solani damping-off of cucumber and plant growth promotion of dry bean (Phaseolus vulgaris L.). In vivo biological control and growth promotion studies were carried out under greenhouse and shadehouse conditions with the use of seed treatment as the method of application. In vitro and in vivo screening was undertaken to select the best Trichoderma isolates from 20 Trichoderma isolated from composted soil. For in vitro screening, dual culture bioassays were undertaken and assessed for antagonisms/antibiosis using the Bell test ratings and a proposed Invasive Ability rating based on a scale of 1-4 for possible mycoparasitic/hyperparasitic activity. The isolates were further screened in vivo under greenhouse conditions for antagonistic activity against R. solani damping-off of cucumber (Cucumis sativus L.) cv. Ashley seedlings. The data generated from the in vivo greenhouse screening with cucumber plants were analysed and grouped according to performance of isolates using Ward‟s Cluster Analysis based on a four cluster solution to select the best isolates in vivo. Isolates exhibiting marked mycoparasitism of R. solani (during ultrastructural studies) viz, T. atroviride SY3A and T. harzianum SYN, were found to be the best biological control agents in vivo with 62.50 and 60.06% control of R. solani damping-off of cucumber respectively. The in vitro mode of action of the commercial Trichoderma product, Eco-T®, and Bacillus B69 and B81 suggested the production of antimicrobial substances active against R. solani. In vitro interaction studies on V8 tomato juice medium showed that the Trichoderma and Bacillus isolates did not antagonise each other, indicating the possibility of using the two organisms together for biological control and plant growth promotion studies. Greenhouse studies indicated that combined inoculation of T. atroviride SYN6 and Bacillus B69 gave the greatest plant growth promotion (43.0% over the uninoculated control) of bean seedlings in terms of seedling dry biomass. This was confirmed during in vivo rhizotron studies. However, results obtained from two successive bean yield trials in the greenhouse did not correlate with the seedling trials. Moreover, no increase in protein or fat content of bean seed for selected treatments was observed. In the biological control trials with cucumber seedlings, none of the Trichoderma and Bacillus combinations was better than single inoculations of Eco-T®, T. atroviride SY3A and T. harzianum SYN. Under nutrient limiting conditions, dry bean plants treated with single and dual inoculations of Trichoderma and Bacillus isolates exhibited a greater photosynthetic efficiency that the unfertilized control plants. Bacillus B77, under nutrient limiting conditions, caused 126.0% increase in dry biomass of bean seedlings after a 35-day period. Nitrogen concentrations significantly increased in leaves of plants treated with Trichoderma-Bacillus isolates. However, no significant differences in potassium and calcium concentrations were found. Integrated control (i.e. combining chemical and biological treatments) of R. solani damping-off of cucumber seedlings proved successful. In vitro bioassays with three Rizolex® concentrations, viz., 0.01g.l-1, 0.1g.l-1 and 0.25g.l-1 indicated that the selected Trichoderma isolates were partly sensitive to these concentrations whereas the Bacillus isolates were not at all affected. In a greenhouse trial, up to 86% control was achieved by integrating 0.1g.l-1 Rizolex® with T. harzianum SYN, which was comparable to the full strength Rizolex® (1g.l-1) application. Irrespective of either a single or dual inoculations of Trichoderma and/or Bacillus isolates used, improved percentage seedling survival as achieved with the integrated system, indicating a synergistic effect. The results presented in this thesis further reinforce the concept of biological control by Trichoderma and Bacillus spp. as an alternative disease control strategy. Furthermore, this thesis forms a basis for Trichoderma-Bacillus interaction studies and proposes that the two organisms could be used together to enhance biological control and plant growth promotion.
Characterization of selected Bacillus isolates exhibiting broad spectrum antifungal activity.
(2004) Tewelde, Teklehaimanot Weldeslasie.; Hunter, Charles Haig.; Beukes, Mervyn.
The genus Bacillus is comprised of Gram-positive, rod-shaped, spore-forming bacteria which are well known for their ability to produce a diverse array of antimicrobial compounds. Ofparticular interest is the ability of certain strains to produce antifungal compounds. Such organisms have the potential for application in agriculture where they can be used as biocontrol agents against selected plant pathogenic fungi. A study was undertaken to further characterize selected Bacillus isolates that exhibit broad spectrum antifungal activity. Dual culture bioassays were used to screen seven selected Bacillus isolates for activity against four plant pathogenic fungi in vitro. All isolates were able to inhibit the pathogens to varying degrees. Two isolates, R29 and B81, were selected for further testing and characterization. Further bioassays were performed on five complex nutrient media which were adjusted to pH S.S and 7, and both incubated at 2SoC and 30°C" respectively. It was found that pH and media composition showed significant influences on the antifungal activities of the isolates tested, but that a SoC temperature difference in incubation temperature did not. Tryptone soy agar was found to give rise to the largest inhibition zones. Both isolates were tentatively identified using standard biochemical and morphological tests. Based on its phenotypic characteristics, R29 was identified as a strain of B. subtilis. B81 proved to be more difficult to assign to a specific group or species of Bacillus, though B. subtilis and B. licheniformis were considered to be the nearest candidates. Genomic DNA was extracted from both isolates and a portion of each of their 16s rDNA genes were amplified and sequenced for homology testing against the GeneBank database. Homology testing confirmed that both isolates were members of the genus Bacillus and most probably strains of B. subtilis. The DNA fragment used for sequencing proved to be too small to give conclusive identification of the isolates. Isolate R29 was selected for further characterization of its antifungal compound/so Growth curve studies using a defined synthetic medium showed that antifungal activity arose during the stationary phase and appeared to be closely linked to sporulation. The antifungal component of cell free culture supematant was extracted using various methods including thin layer chromatography, acid precipitation, hydrophobic interaction chromatography and methanol extractions. High performance liquid chromatography (HPLC) analysis of extracts from acid precipitation and hydrophobic interaction chromatography revealed two active peaks indicating that at least two antifungal compounds were produced. Methanol extracted samples produced the cleanest sample extract but only revealed one active peak from the HPLC fraction . Nuclear magnetic resonance analysis of purified samples indicated that the antifungal compound/s have aromatic complex and peptide structures. The extracted antifungal compounds were Protease K resistant and found to be thermostable at temperatures ranging 80-121oC, and, were active at pH ranges of 3-13. The antifungal compounds were found to exhibit similar properties to known antifungallipopeptides i.e. iturin A and fengycin A and B. Further characterization and identification of the active compounds is recommended usmg methods such as liquid chromatography mass spectrometer and matrix-assisted laser desorption ionisation time-of- flight. The results presented in this dissertation provide a basis from which antifungal compounds produced by strains ofBacillus can be further characterized.
Development of a laboratory river model to determine the environmental impacts of key xenobiotic compounds.
(1996) Hunter, Charles Haig.; Senior, Eric.; Howard, John.; Bailey, Ian.
Microorganisms are increasingly used in toxicological studies to determine potential environmental impacts of xenobiotic compounds. A multi-stage laboratory model was developed to facilitate the examination of environmental impacts of selected pollutants on fundamental cycling processes inherent to aquatic ecosystems, namely, the degradation of organic substances and nitrogen transformations under aerobic conditions. A microbial association representative of riverine ecosystems was enriched for, isolated and cultured within the model. Characterisation of the microbial association were undertaken. Scanning electron microscopy and bright field microscopy revealed that a diverse heterogenous community of microorganisms had established within the model. Successional metabolic events, namely organic carbon catabolism, ammonification of organic nitrogen and the process of nitrification were differentiated in time and space with the microbial association integrity still being retained. The establishment of a microbial association within the model was primarily dependent on: dilution rates, specific growth rates and interactions between microorganisms and the prevailing environmental conditions. Growth-rate independent populations of microorganisms established within the model and were thought to contribute significantly to the metabolic processes within the model. Nitrifying activity was identified as a rate-limiting process within the model. Following separation of metabolic events, the ecotoxicological impacts of phenol and 2,4-dichlorophenol on the association were assessed. The biological oxidation of ammonia through to nitrate (nitrification) was found to be a sensitive indicator of perturbation. The model was found to be suitable for testing both acute and chronic intoxication by pollutant compounds as well as for biodegradation testing and the possible evaluation of ecotoxicological impacts of wastewater treatment plants. The main disadvantages of the model arose from its operational complexity, its empirical nature and its impracticality for screening large numbers of compounds. A bioassay based on the inhibition of ammonium oxidation was developed in order to fulfil the requirements for a simple and rapid test protocol for the initial screening of perturbant compounds.
Diversity of quorum sensing pherotypes amongst ecotypes of plant-associated Bacillus subtilis sensu lato isolates.
(2021) Tredgold, Heather Rayne.; Hewer, Raymond.; Hunter, Charles Haig.
Ecologically-adapted populations, or ecotypes, are species forms that are functionally adapted to particular habitat niches. Bacterial ecotypes are challenging the significance and implications of ecological adaptation in terms of prokaryote taxonomy, community ecology, and biocontrol applications. Plant-associated members of the Bacillus subtilis sensu lato group—in particular B. subtilis and B. velezensis—are ecologically specialised to perform numerous functions that are beneficial to plant and soil health. Underpinning these beneficial activities is the ability to colonise plant surfaces by biofilm formation. Biofilms are a result of co-ordinated social behaviour amongst microorganisms. In the B. subtilis sensu lato this sociality is governed by the ComQXPA quorum-sensing cassette, which uses the ComX pheromone for intercellular communication leading social behaviours like biofilm formation. This peptide pheromone contains a post-translational modification on a conserved tryptophan residue. This modification is highly variable between populations, resulting in discrete ComX variants which form communication groupings known as pherotypes. The limitation of communication to within a pherotype may constitute a form of ecological adaptation designed to protect the products of co-operative behaviour and restrict their benefit to the producing population. The present study aimed to explore ecotypes and pherotypes amongst a subset of plantassociated B. subtilis and B. velezensis isolates. These isolates originated from phylloplane and rhizosphere samples from seven crop species grown across the KwaZulu-Natal province, South Africa, and had demonstrated biocontrol potential in previous studies. An exploratory study set out to apply in silico approaches to determine gene-sequence-based variation amongst representative strains of the B. subtilis sensu lato available in the GenBank database. Nine housekeeping gene targets (viz., 16S rRNA, cheA, dnaJ, groEL, gyrA, gyrB, polC, purH, and rpoB) were evaluated for suitability to resolve clustering of closely-related B. subtilis sensu lato. Four of these gene sequences (viz., 16S rRNA, dnaJ, gyrA, and rpoB) were identified as candidates for a Multilocus Sequence Analysis (MLSA) scheme to distinguish between members of the B. subtilis sensu lato group. Putative pherotype variation amongst these reference strains was explored in silico using comQXP gene sequence data. The suitability of a comQXP PCR-RFLP protocol with potential for rapid pherotyping amongst B. subtilis sensu lato was evaluated in silico using simulated comQXP amplicons. This necessitated the design of a PCR primer set targeting the quorum-sensing gene region of B. velezensis. Four restriction enzyme candidates namely, BtsCI, Fnu4HI, Cac8I, and Hpy166II, were identified for further study. Ecotyping amongst the B. subtilis and B. velezensis isolates was carried out using a four-gene (viz., 16S rRNA, dnaJ, gyrA, and rpoB) MLSA. This concatenated sequence dataset was applied to ecotype simulation (ES) analysis to corroborate putative ecotype sub-clusters in the MLSA phylogeny. Two DNA fingerprinting approaches—Repetitive Element Palindromic PCR (Rep-PCR) and Random Amplified Polymorphic DNA PCR (RAPD-PCR)—were also evaluated for their potential to identify putative ecotypes within the isolate subset. This investigation of pherotypes amongst the putative ecotype groupings examined isolate comQ sequence data as well as the comQXP PCR-RFLP, and also applied a srfA-LacZ reporter gene assay to examine isolate stimulation of seven known pherotype tester strains (viz., 168, RO-B- 2, RO-C-2, RO-E-2, RO-FF-1, RO-H-1, and RS-D-2). The MLSA of isolate gene sequences presented distinct sequence clusters suggestive of ecotype populations amongst the two Bacillus species which were corroborated by ES analysis. The MLSA and ES determined two putative ecotypes within the B. subtilis isolates, and six within the B. velezensis isolates. Ecotype groupings were found to contain isolates from different crop species and locations, and four B. velezensis ecotypes were distinct from included B. subtilis sensu lato reference strains. PCR fingerprinting identified strain-level variances amongst the isolates, and were able to differentiate plant-associated B. velezensis from closelyrelated B. amyloliquefaciens, but did not define groupings entirely consistent with the ES and MLSA phylotypes. The MLSA, ES, and PCR fingerprinting delineated a group of five isolates (viz., B81, CT-R67, bnd136, bng221, and sqo271) to be grouped with reference strain Bacillus sp. JS in the MLSA. This grouping is of interest as Bacillus sp. JS is a strain demonstrating biocontrol capability and formed a clade distinct to B. subtilis sensu stricto in gene sequence phylogenies. This Bacillus sp. JS grouping was further confirmed by ES to constitute a single ecotype, and both Rep-PCR and RAPD-PCR OPG-16 distinguished this grouping based on fingerprint profiles. Investigation of reference strain variation in the comQ gene sequence determined significant levels of sequence variation amongst reference strains evaluated in silico, and showed divergence in some strains from known pherotype counterparts. Furthermore, sequence clusters were resolved in B. velezensis that suggested unique pherotype variants amongst reference strains, and showed comQ similarity amongst the five isolates related to Bacillus sp. JS. This trend was observed for the comQ phylogeny applying isolate and pherotype sequences, which resolved two distinct clusters each amongst the B. velezensis and B. subtilis isolates, with only two isolates (viz., bnd134 and bnd156) grouping with known pherotypes. The B. velezensis clades demonstrated significant comQ sequence variance from the pherotype reference strains based on clustering distances in the phylogeny. Of the four enzyme candidates identified for potential use in a comQXP PCR-RFLP protocol, two were found to be applicable: BtsCI resolved the grouping of the Bacillus sp. JS clade, while Fnu4HI was found to distinguish between more closely-related isolates in B. velezensis. The profile groupings for both of these enzymes demonstrated most of the isolates to be distinct from known reference pherotype strains, and the technique proved able to corroborate groupings in the comQ phylogeny. The pherotyping srfA-LacZ reporter gene assay failed to support the pherotype groupings defined by the comQ phylogeny, but demonstrated that the environmental isolates were capable of eliciting substantial responses in the pherotype tester strains, particularly amongst the B. velezensis isolates. Eight isolates did not elicit significant responses in any of the tester strains, while most matched to multiple pherotypes on the basis of tester strain response to conditioned medium from isolates. These findings demonstrated that putative ecotype variation was present amongst the B. subtilis and B. velezensis isolates that were resolvable by MLSA, and that these ecotype groups comprised isolates from different crop types and location sites. Some of these ecotypes bore only distant relation to B. subtilis sensu lato reference strain counterparts. The investigation of PCR fingerprinting methods for ecotyping purposes found that the primer sets applied were not able to consistently corroborate putative ecotype groupings defined by MLSA and ES. Pherotyping investigations demonstrated that there was evidence of gene-sequence-based pherotype variation amongst the isolates within these putative ecotype groupings. The pherotype clades resolved with the comQ phylogeny and with comQXP PCR-RFLP were not corroborated by srfA-LacZ reporter gene pherotyping assays, but the data suggested that assay optimisation to suit environmental strains is warranted in future studies. This study presents the first instance of ecotyping and pherotyping amongst plant-beneficial B. velezensis. The B. velezensis isolates were largely distinct in ecotype and pherotype from the plant-associated model strain FZB42, which further promotes that these isolates demonstrate localised adaptation. The Bacillus sp. JS-related isolate grouping is of interest as these isolates comprised a single putative ecotype resolvable by MLSA, ES, and Rep-PCR and RAPD-PCR OPG-16. This grouping was distantly-related to B. subtilis sensu stricto, and this is interesting from a taxonomic standpoint, as is the fact that that Bacillus sp. JS is reported to have plantbeneficial capabilities. The existence of uniquely South African plant-associated B. subtilis sensu lato ecotypes is valuable in agricultural management approaches targeting beneficial microbes, as these organisms likely possess adaptations allowing them to compete with extant bacterial communities and thrive within the South African agricultural climate.
The effect of water treatment residues on soil microbial and related chemical properties.
(2003) Pecku, Shantel.; Hunter, Charles Haig.; Hughes, Jeffrey Colin.
Water treatment residue (WTR), a by-product of the water treatment process, consists primarily of precipitated hydroxides of the coagulants used in the water treatment process, along with sand, silt, clay, humic compounds, and dissolved organic matter. It is usually disposed of by landfill, a technology with numerous problems that include dwindling landfill capacity, extensive dewatering requirements for the WTRs, high costs of transportation, and potential liability for landfill clean-up. Therefore, land disposal (or land treatment) presents a popular alternative disposal method based on the principle that the physical, chemical, and microbial properties of the soil can be used to assimilate applied waste without inducing any negative effects on soil quality. The objective of this study was to investigate the effects of land disposal of the WTR generated by Umgeni Water, a local water treatment authority, on soil quality. These effects were investigated using depth samples from soil profiles of Westleigh and Hutton soil forms at field trials located at Ukulinga Research Farm, near Pietermartizburg and Brookdale Farm, Howick, KwaZulu-Natal, South Africa, respectively. Four rates of WTR (0, 80, 320, and 1280Mg ha-1 incorporated into the soil) were investigated at both trials, in addition to mulched treatments at rates of 320 and 1280Mg ha-1 at Brookdale only. Sampling of plots was carried out in September 2001 and May 2002, and all treatments were investigated under fallow and grassed cultivation. Laboratory measurements used to assess soil quality included pH, electrical conductivity (EC), organic carbon (QC), and microbial activity using f1uorescein diacetate (FDA) hydrolysis. At both trials in September 2001 WTR-amended plots displayed higher pH in the 0-200mm soil in comparison to the controls, whereas by May 2002 pH had returned to the condition of the controls. Addition of WTR at Ukulinga resulted in higher QC in September 2001, but in May 2002 this was similar to the controls. However, at Brookdale QC was unaffected by WTR. At Ukulinga and Brookdale the effect of WTR on EC was variable, and microbial activity in the soil profile was unaffected by WTR addition. Observations at Ukulinga and Brookdale reflected long term changes (3 and 5 years, respectively) to soil quality following WTR addition. To examine the initial changes in soil quality a laboratory experiment was set up using the field trial soils. Research objectives were also extended to include WTRs from Rand Water (Johannesburg), Midvaal Water Company (Stilfontein), Amatola Water (East London), and two samples from the Faure Water Treatment Plant (near Cape Town). The second Faure sample (Faure2 ) was collected when blue green algal problems were experienced at the plant. The measurements used to investigate these short term effects on soil quality were soil pH, EC, and microbial activity as indicated by respiration rate. Each of the WTRs added to the Hutton and Westleigh soils increased soil pH by varying increments, and the higher the WTR application rate, the higher was the pH recorded. With the exception of the Rand and Umgeni WTRs that clearly increased soil EC, the effect of the otherWTRs on EC was variable. The Faure1 and Amatola WTRs appeared to have no effect on microbial activity, whereas the Umgeni, Rand, Midvaal, and Faure2 WTRs stimulated microbial activity by Day 2 following the addition of WTR, but this had declined by Day 14. As for pH, higher microbial activity was recorded at higher WTR application rates. Changes in microbial community structure of the Hutton soil only, following the addition of WTR were examined using denaturing gradient gel electrophoresis (DGGE) analysis. Community profiles of the different WTRs proved to be markedly different. However, WTR-amended soil retained banding patterns consistent with the control soil indicating that dominant populations in the Hutton soil had been retained. The field trials indicated that long term effects of land disposal of WTR were not detrimental to the measured indicators of soil quality namely, pH, EC, QC, and microbial activity. The laboratory assessments of the short term response of the Hutton and Westleigh soil forms to WTR addition suggested that the tested variables were altered by WTR, but not significantly changed to the detriment of soil quality. Microbial community analysis indicated that the community structure of the Hutton soil was not significantly altered by WTR amendments. Present findings provide no evidence to suggest that land disposal of WTR is detrimental to soil quality. It is therefore regarded as a feasible disposal option although there are some aspects that should be investigated further. These include investigations into rhizosphere/microbial interactions and the feasibility of growing cash crops.
The effects of land use and management practices on soil microbial diversity as determined by PCR-DGGE and CLPP.
(2011) Wallis, Patricia Dawn.; Titshall, Louis William.; Hunter, Charles Haig.; Morris, Craig Duncan.
The environmental impact of anthropogenic disturbances such as agriculture, on the soil ecosystem, and particularly on soil microbial structural and functional diversity, is of great importance to soil health, conservation and remediation. Therefore, this study assessed the effects of various land use and management practices on both the structural (genetic) and functional (catabolic) diversity of the soil bacterial and fungal communities, at two long-term sites in KwaZulu-Natal. The first site is situated at Baynesfield Estate, and the second at Mount Edgecombe Sugarcane Research Institute. At site 1, the land uses investigated included soils under pre-harvest burnt sugarcane (Saccharum officinarum, Linn.) (SC); maize (Zea mays, Linn.) under conventional tillage (M); permanent kikuyu (Pennisetum clandestinum, Chiov) pasture (KIK); pine (Pinus patula, Schiede) plantation (PF); and wattle (Acacia mearnsii, De Wild) plantation (W), all fertilized; and undisturbed native grassland (NAT) that had never been cultivated or fertilized. At site 2, a sugarcane (Saccharum officinarum × S. spontaneum var. N27) pre-harvest burning and crop residue retention trial was investigated. The treatments studied included conventional pre-harvest burning of sugarcane with the tops removed (Bto), and green cane harvesting with retention of crop residues on the soil surface as a trash blanket (T). Each of these treatments was either fertilized (F) or unfertilized (Fo). The polymerase chain reaction (PCR), followed by denaturing gradient gel electrophoresis (DGGE) were used to determine the structural diversity, and community level physiological profiling (CLPP) using BIOLOG plates, the catabolic diversity. In addition, the soils were analysed with respect to selected physicochemical variables, and the effects of these on the soil microbial communities were determined. Replicate soil samples (0–5 cm) were randomly collected from three independent locations within each land use and management, at both sites. Soil suspensions for the CLPP analyses were prepared from fresh soil subsamples (within 24 h of collection) for the bacterial community analyses, and from 8-day-old soil subsamples (incubated at 4°C to allow for spore germination) for the fungal community analyses. BIOLOG EcoPlates™ were used for the bacterial CLPP study and SF-N2 MicroPlates™ for the fungal analysis, the protocols being adapted and optimized for local conditions. This data was log [X+1]-transformed and analysed by principal component analysis (PCA) and redundancy analysis (RDA). For PCRDGGE, total genomic DNA was isolated directly from each soil subsample, and purified using the MO BIO UltraClean™ soil DNA Isolation kit. Protocols were developed and optimized, and fragments of 16S rDNA from soil bacterial communities were PCR-amplified, using the universal bacterial primer pair 341fGC/534r. Different size 18S rDNA sequences were amplified from soil fungal communities, using the universal fungus-specific primer pairs NS1/FR1GC and FF390/FR1GC. Amplicons from both the bacterial and fungal communities were fingerprinted by DGGE, and bands in the fungal DGGE gels were excised and sequenced. The DGGE profiles were analysed by Bio-Rad Quantity One™ Image analysis software, with respect to band number, position, and relative intensity. Statistical analyses of this data then followed. Soil properties [organic C; pH (KCl); exchangeable acidity; total cations (ECEC); exchangeable K, Ca and Mg; and extractable P] were determined by PCA and were shown to have affected the structural and catabolic diversity of the resident microbial communities. At Baynesfield, canonical correspondence analysis (CCA) relating the selected soil variables to bacterial community structural diversity, indicated that ECEC, K, P and acidity were correlated with CCA1, accounting for 33.3% of the variance, whereas Mg and organic C were correlated with CCA2 and accounted for 22.9% of the variance. In the fungal structural diversity study, pH was correlated with CCA1, accounting for 43.8% of the variance, whereas P, ECEC and organic C were correlated with CCA2, and accounted for 30.4% of the variance. The RDA of the catabolic diversity data showed that the same soil variables affecting fungal structural diversity (organic C, P, ECEC and pH) had influenced both the bacterial and fungal catabolic diversity. In both the bacterial and fungal RDAs, organic C, P and ECEC were aligned with RDA1, and pH with RDA2. However in the bacterial analysis, RDA1 accounted for 46.0%, and RDA2 for 27.5% of the variance, whereas in the fungal RDA, RDA1 accounted for only 21.7%, and RDA2 for only 15.0% of the variance. The higher extractable P and exchangeable K concentrations under SC and M, were important in differentiating the structural diversity of these soil bacterial and fungal communities from those under the other land uses. High P concentrations under M were also associated with bacterial catabolic diversity and to a lesser extent with that of the soil fungal communities under M. Similarly, the higher organic C and exchangeable Mg concentrations under KIK and NAT, possibly contributed to the differentiation of these soil bacterial and fungal communities from those under the other land uses, whereas under PF, the high exchangeable acidity and low pH were possibly influencing factors. Under W, low concentrations of P and K were noted. Other factors, such as the presence/absence and frequency of tillage and irrigation, and the diversity of organic inputs due to the diversity of the above-ground plant community, (in NAT, for example) were considered potentially important influences on the nature and diversity of the various land use bacterial and fungal communities. At Mount Edgecombe, CCA showed that organic C and Mg had a significant effect on soil bacterial structural diversity. Organic C was closely correlated with CCA1, accounting for 58.7% of the variance, whereas Mg was associated with CCA2, and accounted for 41.3% of the variance. In the fungal structural diversity study, ECEC and pH were strongly correlated with CCA1 and accounted for 49.1% of the variance, while organic C was associated with CCA2, accounting for 29.6% of the variance. In the functional diversity studies, RDA showed that both bacterial and fungal community catabolic diversity was influenced by soil organic C, pH, and ECEC. In the bacterial analysis, RDA1 was associated with organic C and pH, and accounted for 43.1% of the variance, whereas ECEC was correlated with RDA2, accounting for 36.9% of the variance. In the fungal analysis, RDA1 was correlated with ECEC and accounted for 47.1% of the variance, while RDA2 was associated with pH and organic C, accounting for 35.8% of the variance. The retention of sugarcane harvest residues on the soil surface in the trashed treatments caused an accumulation of organic matter in the surface soil, which did not occur in the pre-harvest burnt sugarcane. This difference in organic C content was a factor in differentiating both bacterial and fungal communities between the trashed and the burnt treatments. Soil acidification under long-term N fertilizer applications caused an increase in exchangeable acidity and a loss of exchangeable Mg and Ca. Thus, as shown by CCA, a considerably lower exchangeable Mg concentration under F compared to Fo plots resulted, which was influential in differentiating the bacterial and fungal communities under these two treatments. In the structural diversity study at Baynesfield, differences were found in bacterial community species richness and diversity but not in evenness, whereas in the fungal analysis, differences in community species richness, evenness and diversity were shown. The soil bacterial and fungal communities associated with each land use were clearly differentiated. Trends for bacterial and fungal diversity followed the same order, namely: M < SC < KIK < NAT < PF < W. At Mount Edgecombe, no significant difference (p > 0.05) in bacterial structural diversity was found with oneway analysis of variance (ANOVA), but two-way ANOVA showed a slight significant difference in bacterial community species richness (p = 0.05), as an effect of fertilizer applications. A significant difference in fungal species richness (p = 0.02) as a result of management effects was detected, with the highest values recorded for the burnt/fertilized plots and the lowest for the burnt/unfertilized treatments. No significant difference was shown in species evenness, or diversity (p > 0.05), in either the bacterial or the fungal communities. In the catabolic diversity study at site 1, the non-parametric Kruskal-Wallis ANOVA showed that land use had not affected bacterial catabolic richness, evenness, or diversity. In contrast, while fungal catabolic richness had not been affected by land use, the soil fungal community catabolic evenness and diversity had. At site 2, the land treatments had a significant effect on soil bacterial community catabolic richness (p = 0.046), but not on evenness (p = 0.74) or diversity (p = 0.135). In the fungal study, land management had no significant effect on the catabolic richness (p = 0.706), evenness (p = 0.536) or diversity (p = 0.826). It was concluded, that the microbial communities under the different land use and trash management regimes had been successfully differentiated, using the optimized protocols for the PCR-DGGE of 16S rDNA (bacteria) and 18S rDNA (fungi). Sequencing bands produced in the 18S rDNA DGGE, enabled some of the soil fungal communities to be identified. CLPP of the soil microbial communities using BIOLOG plates showed that, on the basis of C substrate utilization, the soil bacterial and fungal communities’ catabolic profiles differed markedly. Thus, it was shown that the different land use and management practices had indeed influenced the structural and catabolic diversity of both the bacterial and fungal populations in the soil.
The establishment of in vitro screening methods for evaluating the susceptibility of sugarcane (Saccharum spp. hybrids) to the fungal disease, smut (causal agent : Ustilago scitaminea H. and P. Sydow) and the stalk borer, Eldana saccharina Walker (Lepidoptera : Pyralidae).
(2010) Devnarain, Natrisha.; Snyman, Sandra Jane.; Hunter, Charles Haig.
The fungal disease smut (causal agent: Ustilago scitaminea H. & P. Sydow) and stalk borer Eldana saccharina Walker place major constraints on sugarcane agriculture in South Africa. The best approach for management is the introduction of resistant cultivars; however, conventional field-based screening for pest and disease resistance is a lengthy process. This study evaluated in vitro techniques combined with artificial inoculation of 12 week old in vitro plantlets and 8-10 week old embryogenic calli as rapid screening methods. Preliminary investigations were conducted on cultivars with known field ratings to smut and E. saccharina: NCo376, N26 and N39; and 5 „test‟ cultivars, whose identities were undisclosed until completion of experiments, were used to assess the accuracy of protocols. Infective U. scitaminea sporidia generated from teliospores, were used as inocula. Development of a callus protocol was unsuccessful due to sporidial and mycelial overgrowth, despite addition of a contact fungicide, Dithane M-45® (0.025 g/l) and a biocide/fungicide, PPMTM (5 ml/l), to media. Plantlet inoculation by injection, 1 cm above the apical meristem, resulted in 12% and 20% of smut susceptible NCo376 plantlets producing smut whips after 5 weeks when inoculated with 1 x 106 and 1 x 109 sporidia/ml, respectively. Smut whip production in 5 of the 8 (63%) cultivars inoculated with the lower sporidial concentration correlated with their field resistance ratings. In addition, whips harvested from in vitro plantlets were a valuable source of aseptic teliospores for future research. Ongoing work involves inoculation of NCo376 calli with such teliospores and maintenance on medium with PPMTM - emergence of whips from plantlets remains to be assessed. The E. saccharina screening protocol involved surface decontamination of eggs with 1% sodium hypochlorite (NaOCl) for 15 min. Feeding bioassays were conducted by placement of first instar larvae on in vitro plantlets and calli for 3 and 2 weeks, respectively. Larval mass, length and percentage infestation were recorded. Although greater larval size was expected in susceptible compared with resistant cultivars, the results did not support this. Significant differences in plantlet infestation were observed between susceptible (94-98%) and resistant (72-86%) lines. No significant differences were found in the callus feeding bioassay. However, a 24 h callus choice bioassay which investigated larval preference between callus genotypes compared with NCo376, showed significant differences and correctly discerned cultivar susceptibility according to field ratings.
Evaluation of growth media parameters for the cultivation of selected biological control agents.
(2002) Phillips, Shivaani.; Laing, Mark Delmege.; Hunter, Charles Haig.
Trichoderma harzianum kmd, Gliocladium virens MMI and Colletotrichum gloeosporioides C6 are potential biological control agents. Trichoderma harzianum kmd and and G. virens MMI have been shown to have excellent growth stimulation and disease suppressive characteristics by the Biocontrol for Africa team of researchers at the University of Natal, Pietermaritzburg. Colletotrichum gloeosporioides C6 has been shown to have effective control of the invasive weed, Hakea sericea (Shrad.). The aim of this dissertation was to establish a method which was most effective for the mass production of the biological control agents (BCAs). Various parameters and the impact of carbon-to-nitrogen and total organic carbon (TOC) on the growth of the BCAs were investigated. Fingerprinting and detection of mutations between strains of Colletotrichum gloeosporioides C6 of different ages were attempted using AFLPs for patenting purposes. Pine shavings and molasses were used in the semi-solid fermentation of T harzianum kmd, G. virens MMI and C. gloeosporioides C6. A 70% (v/v) ethanol soak was the most effective pretreatment in the removal of resin off the pine shavings as well as eradication of contamination. Parameters tested were pH, C:N ratios and TOCs. The optimal pH range for T harzianum kmd and C. gloeosporioides C6 was between pH 6.5 and 7. The optimal pH for G. virens MMI was pH6. Various C:N ratios and TOCs produced highly significant differences in spore yield and mycelial biomass (P
Evaluation of lanthanum/iron oxide amended formable biochar for phosphorous and nitrogen removal in wastewater: preparation, mechanism, and application.
(2022) Sun, Enhui.; Hunter, Charles Haig.; Yang, Lingzhang.
Abstract available in PDF.
Evaluation of the larvicidal potential of Bacillus velezensis strain PHP1601 as a viable biological control agent against selected fly species.
(2024) Ramesar, Danvir Rajesh.; Hunter, Charles Haig.
Flies are one of the most abundant and prevalent insect pests posing a growing threat to various sectors of the economy. In response to this, a study was undertaken to evaluate Bacillus spp. strain PHP1601 as a candidate biocontrol agent against Lucilia cuprina larvae as a proxy for fly species of biocontrol significance. The identity of PHP1601 was confirmed as B. velezensis using MLSA and species-specific PCR. Bioassays demonstrated a larvicidal effect of cell, endospore (102 – 1010 cells/endospores g -1 ) and cell-free supernatant (1 – 30% v w -1 ) treatments on second instar larvae of L. cuprina. Studies were directed to the larvicidal effect of extracellular compounds, namely lipopeptides. Crude lipopeptide extract (CLP) was acquired using organic extraction from Landy broth. Bioassays with CLP extract (5 – 1000 μg g -1 ) resulted in a dose-dependent larvicidal response. Lipopeptides in the CLP extract were purified by TLC and characterised using UPLC ESI-TOF MS. This indicated the presence of iturin, fengycin and surfactin homologues of which, the purified surfactin fraction (Rf 0.91) was the most larvicidal. Bioassays were repeated with commercial surfactin, confirming its larvicidal potency, exhibiting an LC50 of 9.87 μg g -1 at 240 h. Larvae scent choice tests using TSB and MG bioassay medium fermented by PHP1601 showed that resulting VOCs were attractive to fly larvae, which was considered a viable trait of a fly biocontrol agent. CG-MS of the VOCs produced indicated that ketones were the dominant VOC class and, presumably, the major contributor to this larvae attraction effect. Field performance evaluation using pig manure trials demonstrated successful inhibition of several fly species of agricultural and veterinary importance using endospore treatments (105 and 1010 endospores g -1 ) of PHP1601. qPCR and REP-PCR fingerprinting confirmed that PHP1601 could grow in the manure slurries and was amiable to recovery and monitoring. Zebrafish embryo toxicity bioassays of the CLP produced by PHP1601 indicated that they achieved an LC50 of 22.77 µg ml-1, which characterised these metabolites as slightly toxic. Genome mining detected no genes associated with pathogenicity or virulence and presented no apparent pathogenic threat. The investigation demonstrated that B. velezensis PHP1601 is a viable fly biocontrol candidate and constitutes the first report of a B. velezensis antagonist of Brachycera flies.
In vitro and in vivo screening of Bacillus spp. for biological control of Rhizoctonia solani.
(2003) Kubheka, Bongani Petros.; Hunter, Charles Haig.; Laing, Mark Delmege.
The increasing concerns about chemical pesticides that are environmentally hazardous and the continuous development of resistance by palhogens to chemical pesticides have led to this study. Many studies have shown that some Gram-negative bacteria, such as Pseudomonas flouresens, control plant diseases and promote plant growth. In this study Gram positive bacteria, Bacillus sp., were chosen because of their ability to produce endospores. Endospores can be used in stable, dry formulations. The advantage of using endospores is their ability to survive harsh conditions such as droughts and high temperatures, which give a long shelf life to the biological control agent. Bacillus isolates were recovered from the rhizosphere of 12 different crops, and were subsequently screened in vitro for their antimicrobial activity. Of 130 isolates, 87 exhibited antimicrobial activity against the test organisms: Rhizoctonia solani, Pythium sp., Phytophthora cinnamoni, Fusarium sp., and single representatives of Gram negative and Gram positive bacteria, namely, Erwinia carotovora and Staphylococcus aureus respectively. The Bacillus isolates B77, B81 and B69 inhibited all the test organisms investigated, which suggests that they produced broad spectrum antimicrobial compounds or more than one antimicrobial compound. Of the isolates that showed antimicrobial activity, 78 of them did not inhibit Trichoderma harzianum K D, which is a registered biological control agent; indicating their potential for combined application. Selected Bacillus isolates were tested for the biological control of R. solani under greenhouse conditions in wheat, cabbage, tomato, maize, and cucumber seedlings. Bacillus isolates were applied as seed treatments, and the inoculated seeds were planted in R. solani infested speedling trays. Shoot dry weight measurement of seedlings indicated that 12 out of 19 Bacillus isolates showed significantly different shoot dry weight in wheat whereas all the isolates tested in tomato and cucumber gave significantly different shoot dry weight. No significantly different shoot dry weight was obtained for maize or cabbage. Seed emergence findings indicated that none of the Bacillus isolates gave significantly different emergence percentage on wheat, cabbage, tomato, and maize but all of them showed significantly different emergence percentage on cucumber. The results indicate that both the pathogen and the biological control agents exhibited varying levels of specificity on each crop tested. The biological control potential of the best Bacillus isolates was tested on bean and maize crops in the field. Green bean and maize seeds were coated with the selected Bacillus isolates and then sown under field conditions. For each isolate, four replicate treatment plots were established, with and without a R. solani inoculum. Percentage emergence, plant survival levels to harvesting and yield of maize cobs and green beans pods were measured. For all parameters measured the positive and negative controls were not significantly different thereby rendering the results for the entire field study inconclusive. However, Bacillus isolates B77, BII, R5 and R7 improved green bean pod yield and Bacillus Isolate B8I increased maize yield, indicating their potentials as plant growth promoting rhizobacteria (PGPR).
Investigation into the diversity of antifungal aerobic endospore-forming bacteria associated with bulk and crop rhizosphere soil.
(2011) Musoke, Jolly.; Hunter, Charles Haig.
Members of the genus Bacillus are mainly Gram positive, aerobic rod shaped, endospore-forming bacteria that are increasingly being recognised for their ability to promote plant growth and antagonise fungal pathogens. From a biological control perspective, Bacillus spp. strains that produce antifungal compounds are of particular interest. In this study, aerobic endospore-formers were isolated from an undisturbed indigenous grassland soil and screened for antifungal activity and other plant growth promoting traits. Endospore-formers were also isolated from rhizosphere soil associated with the roots of maize, wheat and kale grown in pots containing soil from the same grassland site. Microbial diversity amongst isolates showing antifungal activity was investigated using different molecular fingerprinting methods, namely, intergenic transcribed spacer–PCR (ITS-PCR), random amplified polymorphic DNA-PCR (RAPD-PCR) and 16S rRNA gene amplification and sequencing. Characterization of the active antimicrobial compound(s) associated with selected isolates was also attempted. Prior to isolating from bulk and rhizosphere soils, samples were pre-heated to eliminate heat sensitive vegetative cells. Mean endospore counts were; wheat rhizosphere, Log 6.03 c.f.u g-1 soil; maize rhizosphere, Log 5.88 c.f.u g-1 soil; kale rhizosphere Log 5.90 c.f.u g-1 soil; and bulk soil Log 5.67 c.f.u g-1soil. A total of three hundred and eighty-four isolates were screened for antagonism towards Rhizoctonia solani using dual-culture plate bioassays. Thirty four of the isolates (~9%) mostly isolated from the bulk soil inhibited R. solani at varying degrees. Differences in antimicrobial interactions were apparent in in vitro bioassay; supposedly due to different concentrations and/or types of antimicrobial compounds. Biochemical tests for amylase, cellulase, chitinase, and proteinase activity, siderophore production and inorganic phosphate solubilisation were conducted. None of the isolates possessed all of these attributes and only a few showed multiple traits. Ninety-one percent of the isolates exhibited proteinase activity, 76% were able to hydrolyze starch whereas only four displayed cellulase activity. Only four isolates from the bulk-soil were capable of solubilising inorganic phosphate. ITS-PCR and 16S rRNA gene sequence analysis showed high levels of genetic homology amongst isolates and the majority were closely associated with representatives of the B. cereus group. Isolate C76 was the exception, being closely matched with B. subtilis. ITS-PCR banding profile was useful for distinguishing between species but did not distinguish within species. RAPD-PCR distinguished finer levels of genetic diversity between and within sample sets, with primer OPG-11 showing the greatest levels of heterogeneity. DNA extraction methods and the influence of template DNA dilution were investigated to determine their influence on RAPD-PCR analysis reproducibility. Prominent bands were comparable for crude template- and kit-extracted DNA but slight changes in band intensity and in some instances, additional faint bands were observed. At the highest DNA concentrations tested (7 μg/ml), further bands with molecular weights above 2.5 kbp were apparent. Strict standardization of PCR conditions greatly reduced variability of the RAPD-PCR analysis. Isolates from the different sample sets were screened for the presence of genetic markers associated with the biosynthesis of zwittermicin A, an aminopolyol antibiotic produced by some members of the B. cereus group. In an initial screen only one isolate, W96, yielded PCR amplicons consistent with those previously reported in the literature for the zwittermicin A genes. Later a further sixteen isolates grouped with W96 on the basis of the RAPD-PCR fingerprinting profiles, were screened for the presence of these genes. Of these, only six showed PCR amplification products similar to W96. Sequence homology testing against the GenBank database confirmed the presence of the zwittermicin A genes in these isolates. Isolate W96 was selected for further extraction and characterization of its antifungal compound(s). However, after culturing in various broth media cell free supernatants of W96 failed to show antifungal activity in vitro even when the supernatants were concentrated 20-fold. These findings provide a general overview of the diversity of aerobic endospore-forming bacteria present in an undisturbed indigenous grassland soil that exhibited antifungal activity in vitro and the limited influence tested crop rhizospheres have on this diversity. Combined use of ITS-PCR, 16S rRNA sequencing and RAPD-PCR techniques served as a rapid and effective means of grouping isolates for further investigations of their potential use as biocontrol agents and plant growth promoting rhizobacteria.
Microbiology honours students' conceptual development during a beer brewing teaching learning sequence (TLS)
(2010) Tekane, Rethabile Reginalda.; Anderson, Trevor Ryan.; Hunter, Charles Haig.
Brewing is defined as “the combined processes of preparing beverages from the infusion of sound grains that have undergone sprouting, and the subsequent fermentation of the sugary solution produced, by yeast-whereby a proportion of the carbohydrate is converted to ethanol and carbon-dioxide.” It is a complex process that requires knowledge of concepts from disciplines such as biochemistry, chemistry, engineering, microbiology and physics. The micro-brewery apparatus at the University of KwaZulu-Natal is used by the discipline of microbiology as part of a brewing exercise to introduce students to industrial microbiology with the aim of developing their conceptual understanding of the process. So far, though, no research has been conducted in order to fully establish the effectiveness of this exercise in developing such understanding of the brewing process. The aim, therefore, of this study was to investigate the effectiveness of a micro-brewing Teaching-Learning Sequence (TLS) that incorporates the micro-brewery, for promoting students‟ understanding of the scientific concepts of relevance to the brewing process. The following research questions were addressed: 1) What concepts are essential for understanding the process of beer brewing? 2) Did those students with sound conceptions develop deeper understanding during the TLS? 3) Did students show any conceptual difficulties with the brewing concepts? 4) Did any remediation of such difficulties occur during the TLS? 5) Did students show retention of (mis)understanding two months after the brewing practical? 6) What were students‟ attitudes and motivational levels like during the brewing practical? 7) How well did students rate their experiences of the whole TLS? 8) How well did students‟ motivational levels and their rating of the TLS correlate with any changes in understanding? The study involved ten microbiology honours students subjected to a TLS which consisted of: i) three brewing lectures aimed at introducing students to the brewing process; ii) pre- & post tests including concept mapping tasks aimed at addressing research questions 2, 3 & 4; iii) a brewing practical aimed at facilitating students‟ development of mental models and conceptual understanding of the brewing process and their motivation and attitude to this exercise (addressing question 6 & 8); iv) a group discussion which involved a group tasting session and the evaluation and discussion of each group‟s final beer product; v) semi-structured interviews to establish the source (s) of students‟ difficulties and their retention of knowledge or difficulties (questions 2, 4, & 5 addressed); and vi) an evaluation questionnaire aimed at obtaining student opinion of the TLS (addressing question 7). The data obtained was analyzed via inductive analysis. The results revealed the following brewing difficulties: i) belief that glycolysis reactions are non-consecutively linked chemical reactions which are independent of one another; ii) confusion that whirl-pooling cools the wort; and iii) belief that the final specific gravity value is a measure of the amount of sugars converted to ethanol. Comparison between the pre- & post test responses indicated that some students‟ (B, D & K) conceptual understanding including integrated knowledge of the brewing process improved during the TLS and their brewing difficulties were remediated. In contrast, other students‟ (A, C, E, G, H, J & I) conceptual understanding did not improve during the TLS and their brewing difficulties were not remediated. There was also a positive correlation between student attitudes and motivation towards the brewing practical and the quality of their learning outcomes. Students (B, D & K) who showed high motivational levels and cognitively and physically took part in the TLS showed improved conceptual understanding of the brewing process and retention of knowledge, while those showing low motivational levels did not improve. Furthermore, there are students (G, H & J) who showed high motivational levels during the TLS but their conceptual understanding of the brewing process did not improve. The results obtained suggest that the TLS, based on the micro-brewery apparatus, was at least partially effective in facilitating the development of students‟ conceptual understanding and visualization of the brewing process and the remediation of some of their difficulties, which in some case correlated well with their motivational levels and attitudes towards the brewing exercise. More research is however required to fully confirm the usefulness of such TLSs in brewing education.
Revival and characterization of aerobic endospore-forming bacteria from an ancient sediment core obtained from the Mfabeni Peatland, South Africa.
(2017) Naidoo, Selisha.; Hunter, Charles Haig.
Abstract available in PDF file.
Screening for aerobic endospore-forming bacteria as biocontrol agents for powdery mildew disease of cucurbits.
(2015) Tredgold, Heather Rayne.; Hunter, Charles Haig.
Powdery mildew of cucurbits costs the South African cucurbit-growing industry millions of Rands per year in reduced yields and compromised fruit quality. Amongst the many bacterial and fungal antagonists of cucurbit powdery mildew, certain aerobic endospore-forming bacteria (AEFB) species show promise as biocontrol agents of this disease. When embarking upon biocontrol agent selection, multifaceted screening strategies are crucial. A study was undertaken with the aim of isolating AEFB from the cucurbit phylloplane for evaluation as potential antagonists of cucurbit powdery mildew using various screening approaches. Three hundred and nine AEFB isolates were isolated from cucurbit leaf material sourced from eight locations in the greater Msunduzi, KZN region. Dual-culture antifungal bioassays were performed using surrogate phytopathogenic fungi Botrytis cinerea and Rhizoctonia solani in place of the obligately biotrophic Podosphaera spp.. Two PCR-based genotyping methods were used to differentiate and group 55 antifungal AEFB isolates: internal-transcribed spacer region (ITS) PCR and randomly amplified polymorphic DNA (RAPD) PCR. The RAPD-PCR distinguished greater levels of genetic polymorphisms amongst isolates than did the ITS-PCR, revealing 14 different profiles as opposed to the three obtained from ITS-PCR; with 42% of isolates associated with a single RAPD-PCR banding profile. Phylogenetic relationships between representatives of each of the RAPD-PCR fingerprint groupings were determined by sequence analysis of 16S rRNA and gyrase subunit A (gyrA) gene fragments. In each instance, several distinct clusters were discernable, though gyrA sequences displayed higher levels of strain-level sequence heterogeneity. Comparisons of both gene sequence types with reference strains from the GenBank database revealed similarities to several known plant-associated strains of AEFB, including B. amyloliquefaciens subsp. plantarum and B. subtilis. Matrix-assisted laser deionisation-desorption time-of-flight mass spectrometry (MALDI-TOF-MS) based identification of selected AEFB was evaluated by comparing spectral data from AEFB isolates with reference strains in a Bruker BDAL Biotyper database. Only three out of the 14 isolates evaluated were identified to species level with acceptable confidence levels. This poor taxonomic resolution was ascribed to a paucity of applicable reference strains in the BDAL library. Nevertheless, mass spectra profiles of each isolate allowed for the clustering of related isolates to be achieved when dendograms were created. Antifungal compounds were extracted from 14 isolates using an acid-precipitation and methanol extraction protocol. Detection and identification of lipopeptide compounds in these extracts was assessed using thin-layer chromatography (TLC) and MALDI-TOF-MS. PCR-based screening for lipopeptide production potential using selected lipopeptide gene markers (viz. surfactin, iturin, bacillomycin, and fengycin) was also evaluated for the selected 14 isolates. These isolates were found to produce multiple lipopeptide compounds; including homologues of surfactin, iturin, and fengycin. However, disparities that emerged between PCR, TLC, and MALDI-TOF-MS data suggest that some PCR primers, the ituD marker in particular, showed limited specificity amongst the AEFB strains screened. Based on the overall findings, nine isolates proceeded to in vivo screening against Podosphaera spp. using an agarised detached cotyledon assay and a biocontrol pot trial. Isolates achieving the most effective antagonism of Podosphaera spp. differed in each respective assay. Isolate cce175 provided the highest antagonism in the biocontrol pot trial, and isolate sqo279 provided the best results in the detached cotyledon assay. The impacts of inoculum preparation were assessed using isolate cce175 in a biocontrol pot trial. Treatments varied in cell growth phase and assessed cell-free supernatant, whole broth, and cell-only fractions on biocontrol efficacy compared to a Tebuconazole (430 g/l) fungicide control. None of the treatments were found to impact disease at a statistically significant level. The merits and limitations of the various screening approaches used, and issues surrounding the isolation and assessment of biocontrol efficacy in plant-associated AEFB, are discussed.
Screening for biosurfactant production amongst aerobic endospore forming bacteria isolated from Mfabeni peatland sediment core.
(2019) Adu, Folasade Abimbola.; Hunter, Charles Haig.
Abbstract available in pdf.
Screening of aerobic endospore-forming bacterial isolates as candidate biocontrol agents against rhizoctonia solani.
(2016) Hunter, Charles Haig.; Schmidt, Stefan.; Laing, Mark Delmege.; Wallis, Frederick Michael.
Bacterial-based biocontrol of soil-borne phytopathogens has gained prominence as a promising technology for developing sustainable agricultural pest control practices. Aerobic endospore-forming bacteria are seen as potential candidates for biocontrol applications due to various ecological and physiological traits which have been shown to influence plant health and disease control. Their ability to produce endospores also provides a major commercial advantage over non spore-forming bacteria. Appropriate screening methods are central to the discovery of successful biocontrol agents and should ideally be both ecologically relevant and able to evaluate a large number of isolates. A study was therefore undertaken with the aim of establishing screening methods that facilitate the selection of aerobic endospore-forming bacteria as candidate biocontrol agents against Rhizoctonia solani, an economically important fungal pathogen exhibiting a wide host range. Aerobic endospore-forming bacteria were isolated from rhizosphere material of five crop types grown in composted pine bark medium and screened for R. solani antagonism using traditional in vitro dual-culture bioassays. Isolates exhibiting antifungal activity were then evaluated in vivo for biocontrol activity against R. solani in cucumber seedling trials. Selected isolates were evaluated further using several screening approaches including: genomic fingerprinting; characterization of, and PCR-based screening for genes involved in the biosynthesis of bioactive lipopeptide compounds; and, the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a means of rapidly screening bacterial isolates. Approximately 6% of the bacterial isolates (n=400) showed antagonism towards R. solani in vitro. Dual-culture bioassays against R. solani, Fusarium oxysporum, Botrytis cinerea and Pythium arrhenomanes revealed that the antagonistic activity amongst isolates varied considerably and was influenced by the duration of the assay. From these assays it was possible to rank isolates based on the extent and stability of the inhibitory response in vitro as well as by the spectrum of antifungal activity observed. Twenty-four isolates were selected for in vivo screening for biocontrol activity against R. solani, using susceptible cucumber plants grown under greenhouse conditions. In preliminary experiments the pathogen loading rates were shown to have a marked influence on disease severity. In experiments where R. solani was seeded in the form of colonized agar plugs, significant differences between treatments and controls were recorded and several potential biocontrol candidates were identified. A general observation was that isolates that achieved high rankings in vitro performed better in the in vivo trial than those with lesser rankings; although some exceptions were noted. These findings support the notion that fungal antagonism is an important determinant of biocontrol potential that can be used in preliminary biocontrol screening programmes. Internal-transcribed spacer region (ITS) PCR and randomly amplified polymorphic DNA (RAPD) PCR were evaluated as methods to differentiate isolates exhibiting antifungal activity in vitro. ITS-PCR distinguished three major groupings, but proved to be limited in its ability to detect inter- and intra-specific variation amongst closely related organisms. Based on 16S rRNA gene sequence analysis, two of the groups were identified as members of the “Bacillus subtilis” and “Bacillus cereus” clusters; while, the third group consisted of a single isolate identified as a strain of Brevibacillus laterosporus. RAPD-PCR revealed further levels of genetic diversity within each ITS grouping. The “Bacillus subtilis” cluster was distinguished further into four distinct groups, which based on gyrA gene fragment sequence analysis, were identified as strains of B. amyloliquefaciens subsp. plantarum and B. subtilis respectively. Sequence matches were consistent with the RAPD-PCR groupings, indicating that this method was appropriate for differentiating related isolates at the strain and possibly the sub-species level. Clonal similarities were evident for a number of strains isolated from different plant species suggesting that these may reflect populations of rhizosphere competent strains and/or plant adapted ecotypes. Strains of B. amyloliquefaciens subsp. plantarum and B. subtilis were amongst the best performers in the in vivo biocontrol seedling trial and generally performed better than the “Bacillus cereus” group of isolates. RAPD-PCR of the “Bacillus cereus” isolates showed that they exhibited greater levels of genetic heterogeneity and that the groupings detected were not consistent when different primer sets were evaluated. Genomic fingerprinting was found to provide an insight into the prevalence, distribution and possible rhizosphere competency of related strains. Liquid chromatography was used in conjunction with electrospray-ionization time-of-flight (ESI-TOF) mass spectrometry (MS) to characterize bioactive lipopeptides purified from culture supernatants of selected strains that ranked highly in the in vitro/in vivo assays. Phylogenetically related strains produced very similar lipopeptide profiles. Bacillus subtilis strains were found to produce isoforms of surfactin and fengycin. In addition to these lipopeptides, B. amyloliquefaciens subsp. plantarum strains were also found to produce isoforms of bacillomycin D or iturin A. Bacillomycin/iturin and fengycin fractions exhibited antifungal activity in vitro, whereas surfactin fractions did not. Isolates that ranked the highest in the R. solani dual-culture bioassays all produced either isoforms of bacillomycin D or iturin A. Bacillomycin D producing isolates were amongst the best performers in the in vivo biocontrol trials. Gene markers targeting the biosynthetic apparatus of the detected lipopeptide classes were then assessed for screening purposes using PCR. BACC1F/1R primers targeting the bacillomycin D synthetase C (bmyC) gene correlated well with the ESI-TOF MS findings, whereas ITUD1F/1R primers targeting the malonyl-CoA-transacylase (ituD) gene linked to iturin A biosynthesis were unable to distinguish between isolates that produced iturin or bacillomycin in culture. Disparities between some of the PCR and ESI-TOF MS results suggested that primers targeting srfA (surfactin) and fenD (fengycin) biosynthetic genes showed limited specificity amongst the strains screened. Phylogenetic comparisons of srfD and fenD gene sequences from selected strains of B. amyloliquefaciens subsp. plantarum and B. subtilis revealed that these genes clustered according to species with marked heterogeneity between clusters being evident. Using fenD gene sequence data from B. amyloliquefaciens subsp. plantarum FZB42, primers (FENG1F/1R) targeting fengycin synthetase genes of strains of B. amyloliquefaciens subsp. plantarum isolated in this study were successfully established. MALDI-TOF MS was assessed as a means of identifying isolates antagonistic to R. solani in vitro and determining their associated lipopeptide profiles. Mass spectra were obtained in the m/z range 2000 to 20000 for identification and grouping purposes and in the m/z 750 to 2500 range in order to profile lipopeptide production. The available Bruker BDal spectral library allowed for the identification of isolates to the genus level but proved to be limited for identifying environmental isolates to the species level. Extension of the library using “inhouse” mass spectra generated from isolates identified in this study significantly improved the level of isolate identification in subsequent identification runs. Cluster analysis of mass spectra allowed for the relationships between isolates to be established and provided a means of grouping closely related isolates. Strains of B. subtilis and B. amyloliquefaciens were clearly distinguished from one another and the potential for differentiating strains at the subspecies level was also shown. MALDI-TOF MS also provided a convenient means of detecting bioactive lipopeptides directly from whole cell preparations, cell extracts and crude culture filtrates. Lipopeptide profiles varied depending on taxonomic groupings. Results for isolates within the “Bacillus subtilis” group supported the earlier ESI-TOF MS findings and were found to be more reliable than PCR screening for lipopeptide synthesis genes. “Bacillus cereus” group isolates produced distinct spectral profiles with peaks that were consistent with biomarkers previously described in the literature as isoforms of the kurstakin class of lipoheptapeptides. Brevibacillus laterosporus CC-R4 yielded a unique spectral profile in the m/z 750-2000 range with mass fragments which were similar to antimicrobial compounds recently reported in the literature. Overall, MALDI-TOF MS was found to fulfil the requirement for a practical yet robust technique suitable for processing large numbers of aerobic endospore-forming bacteria for biocontrol screening. This study has shown that genomic fingerprinting and MALDI-TOF MS characterization of bacterial isolates are worthwhile additions to preliminary in vitro screening practices. They provide a level of isolate differentiation and characterization that is beneficial for selecting candidate biocontrol agents, which is not possible with traditional screening practices. Effectively, they allow traditional biocontrol screening to move away from empirically based approaches to ones which are “knowledge” based, allowing for representative groups of bacteria with specific traits to be selected for further evaluation.
South African photorhabdus spp. : genetic and antibiotic diversity.
(2016) Van Wyngaard, Matthew George Dennis.; Hunter, Charles Haig.
As antibiotic producing symbionts of entomopathogenic nematodes, members of the genus Photorhabdus are candidates for biological control of insects and phytopathogenic microorganisms. The aim of this project was to establish the levels of genetic and antibiotic diversity amongst twenty Photorhabdus sp. isolates acquired from the South African Small Grain Institute, Agricultural Research Council (ARC) culture collection. These isolates represent a subset of Photorhabdus sp. isolates obtained from the Freestate and Western Cape provinces of South Africa. Bacterial strain diversity was investigated using several techniques; which included phenotypic testing, genomic and proteomic fingerprinting approaches. Phenotypic characteristics were assayed using API 20E biochemical test strips in conjunction with several supplementary tests. Genomic fingerprinting involved 16S rRNA gene PCR-RFLP and RAPD-PCR. Phylogenetic relationships between the isolates were determined using partial 16S rRNA gene sequence analysis. MALDI-TOF-MS was used to generate proteomic profiles for isolate identification and mass spectra comparison. Species-level differentiation between isolates and reference strains was achieved using 16S rRNA gene PCR-RFLP and partial 16S rRNA gene sequence analyses. Higher levels of strain differentiation between isolates were detected by RAPD-PCR and with MALDI-TOF-MS. In comparison to the high resolution of isolate diversity achieved by DNA-based analysis methods, the phenotypic characteristics proved to be of limited value. All methods evaluated suggest that the isolates are closely-related strains of P. luminescens. Two isolates were found to be non-Photorhabdus and identified as strains of Xenorhabdus sp. and Pseudomonas sp.. Antimicrobial activity amongst the isolates was screened for using disc-diffusion bioassays against Escherichia coli, Micrococcus luteus, Bacillus subtilis, Rhizoctonia solani and Botrytis cinerea. All isolates showed bioactivity against the Gram positive test organisms with weaker and variable antagonism against both fungal species. Three representative isolates were taken forward for further analysis of bioactive compounds produced. Antibiotic extraction and purification was attempted using several techniques including liquid-liquid extraction of broth supernatant using ethyl acetate, methanol extraction of cell pellet material and methanol extraction of lyophilised broth. Analysis of crude and partially purified antibiotic extracts was performed using C18 Sep-Pak clean-up, TLC, UPLC-ESI-TOF-MS and GC-MS. All antimicrobial extraction methods yielded bioactive fractions. UPLC-ESI-TOF-MS proved the most valuable analytical method with data obtained suggesting that each isolate produced identical compounds. The dominant UPLC peak for all isolates and extraction procedures displayed an ESI-TOF-MS mass spectrum consistent with the antibiotic 3,5-dihydroxy-4-isopropyistilbene. These results show that very limited genetic and phenotypic diversity existed between isolates. All isolates were identified as members of P. luminescens but did not cluster closely with any previously described subspecies on the basis of 16S rRNA partial gene sequence analysis. Likewise antimicrobial compound diversity between the three isolates assessed was found to be low, with the major bioactive compound identified as one previously described from Photorhabdus spp..